Analysis of different forms of recombinant human leukocyte interferons and synthetic fragments by high-performance liquid chromatography

Identification of an oxidative refolding intermediate of recombinant consensus interferon from inclusion bodies and design of a two‐stage strategy to promote correct disulfide‐bond formation

“…In the present study, by monitoring the folding process of reduced denatured inclusion bodies of rIFN‐con 1 , the SM of rIFNα was clearly demonstrated to be a dynamically trapped intermediate. MALDI–TOF mass spectra of the tryptic digestion mixture of the intermediate indicated that it comprised only one disulfide bond, Cys 29 –Cys 139 , which is consistent with SM reported [12,13]. CD and fluorescence spectra of the intermediate similar to those of the native rIFN‐con 1 indicated that the intermediate formed a highly native‐like structure.…”

“…It was found that this intermediate moved more slowly than the native structure. The same characterization of the intermediate as SM that had been discovered in some purified products of rIFNα [12,13] suggested that SM might also be a dynamically trapped species. In non‐reducing SDS/PAGE, the disulfide bonds in the sample were intact because reducing agent was absent during the analysis process.…”

“…The latter disulfide bond has been confirmed to be essential for antiviral biological activity [11]. It was reported that rIFNα purified by monoclonal antibody affinity chromatography contained two different monomer forms, namely SM (slow‐moving monomer) and FM (fast‐moving monomer), according to their migrating speed on non‐reducing SDS/PAGE [12, 13]. Preparative RP‐HPLC (reversed‐phase HPLC) was used to separate these two forms.…”

Chromatographic Techniques for the Characterization of Proteins

“…Different forms of recombinant human interferons (rhINF) were analyzed on a reversed system (Felix et al, 1985). The method was able to separate rhIFN-aA, rhIFN-aD, and the hybrid rhIFN-aAJD.…”

A simple and rapid HPLC method for quantitation of interferon-α2b in dosage forms and delivery systems

“…In addition, these methods are capable of being used during the production/formulation processes for monitoring of minor structural as well as conformational variations occurred in protein structure which can lead to significant changes in biological activity of the drug [15]. Most of the chromatographic methods presented for IFN-␣2b analysis have gain limited attention in practical use because of a series of inherent problems including very long run-time [13,[16][17][18][19], the need for special columns and/or other equipments, high variations in responses and, finally, the high expertise needed for use and interpretation of the results obtained from these methods [13,[18][19][20][21][22]. Therefore, in this article we have developed and validated a simple and available gradient reversed-phase HPLC method with UV detection for quantitation of IFN-␣2b in parenteral dosage forms and delivery systems.…”